Edta free protease inhibitor cocktail

Manufactured by Promega

Sourced in United States

EDTA-free protease inhibitor cocktail is a versatile solution designed to prevent the degradation of proteins during sample preparation and analysis. It effectively inhibits a broad spectrum of serine, cysteine, and metalloproteinases without the presence of EDTA.

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Lab products found in correlation

Phosstop, by Roche (4 mentions) Bca assay, by Thermo Fisher Scientific (3 mentions) Sep pak, by Waters Corporation (3 mentions) Lys c, by Fujifilm (3 mentions) Trypsin, by Promega (3 mentions) Swiss webster mice, by Jackson ImmunoResearch (1 mentions) Flag peptide, by Merck Group (1 mentions) Anti flag m2 affinity gel, by Merck Group (1 mentions) Microbca reagent, by Thermo Fisher Scientific (1 mentions) Rnasein, by Promega (1 mentions) Whatman, by Cytiva (1 mentions) Sodium dodecyl sulfate (sds), by Thermo Fisher Scientific (1 mentions) Glycerol, by Merck Group (1 mentions) Mercaptoethanol, by Merck Group (1 mentions) Tween 20, by Thermo Fisher Scientific (1 mentions) G5516, by Merck Group (1 mentions) β mercaptoethanol, by Merck Group (1 mentions)

Spelling variants of this product

Protease inhibitor cocktail (139 citations) Protease inhibitors (55 citations)

8 protocols using edta free protease inhibitor cocktail

Murine Tissue Fractionation and Proteomic Analysis

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Five murine brains
and livers were harvested from CO₂ asphyxiated 3-week-old
male Swiss-Webster mice (Jackson Lab, Bar Harbor, ME). Saccharomyces
cerevisiae cells were grown to an OD of 1.0, washed with
ice cold PBS and snap frozen in liquid N₂ until further
use. Brain/liver tissues and yeast cells were mechanically lysed with
a homogenizer in SDS lysis buffer [2.0% SDS w/v, 250 mM NaCl, PhosSTOP
(Roche, Madison, WI) phosphatase inhibitors, 2 mM sodium vanadate,
EDTA free protease inhibitor cocktail (Promega, Madison, WI), and
50 mM HEPES, pH 8.5]. Lysates were reduced with 5 mM DTT and cysteine
residues alkylated with iodoacetamide (14 mM) in the dark (30 min).
Protein was extracted by methanol–chloroform precipitation
and subsequent ice cold acetone washes. Pellets were dried and resuspended
in 8 M urea containing 50 mM HEPES (pH 8.5). Protein concentrations
were measured by BCA assay (Thermo Scientific, Rockford, IL) prior
to protease digestion. Protein lysates were diluted to 4 M urea and
digested with LysC (Wako, Japan) in a 1/200 enzyme/protein ratio overnight.
Protein extracts were diluted further to a 1.5 M urea concentration,
and trypsin (Promega, Madison, WI) was added to a final 1/250 enzyme/protein
ratio for 6 h at 37 °C. Digests were acidified with 200 μL
of 20% formic acid (FA) to a pH ∼2 and subjected to C18 solid-phase
extraction (SPE) (Sep-Pak, Waters, Milford, MA).

Erickson B.K., Jedrychowski M.P., McAlister G.C., Everley R.A., Kunz R, & Gygi S.P. (2014). Evaluating Multiplexed Quantitative Phosphopeptide Analysis on a Hybrid Quadrupole Mass Filter/Linear Ion Trap/Orbitrap Mass Spectrometer. Analytical Chemistry, 87(2), 1241-1249.

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Purification of 46R-3Flag Proteins

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To purify 46R-3Flag associated proteins, 2 × 10⁸ GSTC cells were infected with ADRV_46R-3Flag or wild type ADRV at an MOI of 1. Cells were collected at 24 hpi and resuspended in lysis buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 0.5% NP-40, and 1 mM DTT) with EDTA-free protease inhibitor cocktail (Promega, USA). The cell lysates were centrifuged at 12,000×g for 15 min at 4 °C, and the supernatants were incubated with anti-FLAG M2 affinity gel (Sigma, USA) for 3 h at 4 °C. Then, the gel was washed four times with wash buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, 0.1% NP-40, and 1 mM DTT) and equilibrated with elution buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 1.5 mM MgCl₂, and 1 mM DTT). The bead-bound proteins were eluted with 3× Flag peptide (Sigma, USA). The eluted proteins were analyzed with MS as described above. The assays were performed in triplicate.

Ke F., Yu X.D., Wang Z.H., Gui J.F, & Zhang Q.Y. (2022). Replication and transcription machinery for ranaviruses: components, correlation, and functional architecture. Cell & Bioscience, 12, 6.

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Yeast Proteome Extraction and Digestion

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Saccharomyces cerevisiae cells were grown to an optical density (OD) of 1.0, washed with ice cold PBS, and snap frozen in liquid N₂ until further use. Yeast cells were mechanically lysed with a homogenizer in a SDS lysis buffer [2.0% SDS w/v, 250 mM NaCl, PhosSTOP (Roche, Madison, WI) phosphatase inhibitors, EDTA free protease inhibitor cocktail (Promega, Madison, WI), and 50 mM HEPES, pH 8.5]. Lysates were reduced with 5 mM DTT, and cysteine residues were alkylated with iodoacetamide (14 mM) in the dark (30 min). Protein content was extracted by methanol-chloroform precipitation and subsequent ice-cold acetone washes. Protein pellets were dried and resuspended in 8 M urea that contained 50 mM HEPES (pH 8.5). Protein concentrations were measured by BCA assay (Thermo Scientific, Rockford, IL) prior to protease digestion. Protein lysates were diluted to 4 M urea and digested with LysC (Wako, Japan) in a 1/200 enzyme/protein ratio overnight. Protein extracts were diluted further to a 1.5 M urea concentration, and trypsin (Promega, Madison, WI) was added to a final 1/250 enzyme/protein ratio for 6 h at 37 °C. Digests were acidified with 200 μL of 20% formic acid (FA) to a pH ~2 and subjected to C18 solid-phase extraction (SPE) (Sep-Pak, Waters, Milford, MA).

Erickson B.K., Schweppe D.K., Yu Q., Rad R., Haas W., McAlister G.C, & Gygi S.P. (2020). Parallel Notched Gas-Phase Enrichment for Improved Proteome Identification and Quantification with Fast Spectral Acquisition Rates. Journal of proteome research, 19(7), 2750-2757.

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Mitochondrial Proteomics from Differentiated Cells

Cited 1 time

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Mitochondria were isolated using a previously described protocol (Wieckowski et al., 2009 (link)) using differentiated cells from 12×15 cm plates. Proteomics was performed as previously described(Balsa et al., 2019 (link)). Mitochondrial pellets were solubilized in SDS lysis buffer (4.0 % SDS w/v, 250 mM NaCl, PhosStop (Roche) phosphatase inhibitors, EDTA free protease inhibitor cocktail (Promega), and 50 mM HEPES, pH 8.5), reduced with 5 mM TCEP at 60°C for 30 minutes, alkylated with 14 mM iodoacetamide for 45 minutes, and precipitated using trichloroacetic acid at a final concentration of 25%. Samples were centrifuged and pellets were washed three times with cold methanol. Proteins were resuspended in EPPS buffer and digested with Lys-C (1:100) for 4 hours at 37° C following trypsin digestion (1:100) at 37° C overnight. Peptides were quantified by microBCA reagent (Thermo Fisher Scientific) and TMT labeled (6–8M excess). Formic acid was added to a final concentration of 1% and peptides were clean up using a 50 mg Seppak column. Eluted peptides were dried down and resuspended in 5% ACN/5% formic acid buffer. Ratios were checked by HPLC and samples fractionated. Fractions were resuspended in 1% formic acid and cleaned up using C18-membrane stage tips. Samples were dried down and MS analysis was performed as previously described (Balsa et al., 2019 (link)).

Latorre-Muro P., O’Malley K.E., Bennett C.F., Perry E.A., Balsa E., Tavares C.D., Jedrychowski M., Gygi S.M, & Puigserver P. (2021). A Cold Stress Inducible PERK/OGT Axis Controls TOM70-Assisted Mitochondrial Protein Import and Cristae Formation. Cell metabolism, 33(3), 598-614.e7.

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CLIP Assay for Protein-RNA Interactions

Cited 2 times

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Cross-linking immunoprecipitation (CLIP) assays were as described in Ule et al. [29 (link)]. Briefly, cells or isolated mitoplasts were UV-irradiated on ice, harvested and lysed in Sigma FLAG lysis buffer adjusted with 0.1% SDS (v/v), Roche EDTA-free Protease Inhibitor Cocktail and Promega RNaseIn. Specific RNP complexes were immunoprecipitated. RNA species bound to the protein of interest were dephosphorylated, ligated to the 3′-RNA linker and end-labelled with γ³²P. Protein–RNA complexes were resolved on SDS–PAGE, transferred to nitrocellulose (BA-85 Whatman) and subjected to autoradiography. Appropriately sized RNP complexes were excised and proteinase K-treated. Following ligation of a 5′ RNA linker RNA, CLIP tags were amplified by RT-PCR, and then cloned, sequenced and analyzed as described in ref. [14 (link)], or IonTorrent-sequenced as described in ref. [30 (link)].

Rozanska A., Richter-Dennerlein R., Rorbach J., Gao F., Lewis R.J., Chrzanowska-Lightowlers Z.M, & Lightowlers R.N. (2017). The human RNA-binding protein RBFA promotes the maturation of the mitochondrial ribosome. Biochemical Journal, 474(13), 2145-2158.

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Protein Extraction and Quantification

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Proteins were extracted using a protein lysis buffer
containing 1M NaCl, 10 mM Tris-Cl (pH=7.5), 10%
glycerol (Sigma-Aldrich, USA), 1% Tween^TM20(Affymetrix,
USA), 10 Mm ß-mercaptoethanol (Sigma-Aldrich, USA)
and 1x EDTA-free Protease Inhibitor Cocktail (Promega,
USA). Lysates were sonicated, denatured at 95°C and
diluted in 1x sodium dodecyl sulfate 20 % (Fisher
Scientific, UK). The Coomassie Plus (Bradford) protein
assay (Thermo Scientific, USA) was used to measure the
protein concentration.

Minaidou A., Nicolaou P, & Christodoulou K. (2018). LRSAM1 Depletion Affects Neuroblastoma SH-SY5Y Cell Growth and Morphology: The LRSAM1 c.2047-1G>A Loss-of-Function Variant Fails to Rescue The Phenotype. Cell Journal (Yakhteh), 20(3), 340-347.

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Protein Extraction and Digestion

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Cell pellets were combined 1:1 with SDS lysis buffer (4.0 % SDS w/v, 250 mM NaCl, PhosStop (Roche) phosphatase inhibitors, EDTA-free protease inhibitor cocktail (Promega) and 50 mM HEPES, pH 8.5). Extracts were reduced with 5 mM DTT (57 °C for 30 min) and cysteine residues alkylated with iodoacetamide (14 mM) in the dark (45 min). Extracts were purified by methanol–chloroform precipitation and subsequent ice cold acetone washes. Pellets were resuspended in 8 M urea containing 50 mM HEPES, pH 8.5, and protein concentrations were measured by BCA assay (Thermo Scientific) prior to protease digestion. A total of 200 µg of protein extracts were diluted to 4 M urea and digested with LysC (Wako) in a 1/200 enzyme/protein ratio overnight. Digests were diluted further to a 1.5 M urea concentration and trypsin (Promega,) was added to a final 1/250 enzyme/protein ratio for 6 h at 37 °C. Digests were acidified with 20 µL of 20% formic acid (FA) to a pH ~2 and subjected to C18 solid-phase extraction (SPE) (50 mg, Sep-Pak, Waters).

Soustek M.S., Balsa E., Barrow J.J., Jedrychowski M., Vogel R., Jan S.m.e.i.t.i.n.k., Gygi S.P, & Puigserver P. (2018). Inhibition of the ER stress IRE1α inflammatory pathway protects against cell death in mitochondrial complex I mutant cells. Cell Death & Disease, 9(6), 658.

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Protein Extraction and Quantification

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Proteins were extracted using a protein lysis buffer, that contained 1M NaCl, 10mM Tris-Cl (pH = 7.5), 10% glycerol (G5516, Sigma-Aldrich, U.S.A), 1% Tween20 (Affymetrix, U.S.A.), 10Mm β-Mercaptoethanol (Sigma-Aldrich, U.S.A) and 1x EDTA-free Protease Inhibitor Cocktail (Promega, U.S.A.). Cell lysates were centrifuged at 4°C and supernatants were collected. Lysates were sonicated and denatured at 95°C. Then proteins were diluted in 1x Sodium Dodecyl Sulfate 20% (ThermoFisher Scientific, U.K.). The Coomassie Plus (Bradford) protein assay (23236, ThermoFisher Scientific, U.K.) was used to determine the protein concentration.

Minaidou A., Nicolaou P, & Christodoulou K. (2019). Deregulation of LRSAM1 expression impairs the levels of TSG101, UBE2N, VPS28, MDM2 and EGFR. PLoS ONE, 14(2), e0211814.

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